Adverse drug reactions (ADRs) are a major obstacle for the development of new medicines. They are also a leading cause of patient morbidity and mortality. Although ADRs affect many different organs and bodily systems, drug induced liver injury has lead to the withdrawal of several drugs at the post licensing stage, and is a key cause of drug attrition. Many of the drugs that cause liver injury are thought to do so through metabolism to a reactive metabolite, exposure to which can cause modification of cellular proteins, leading to loss of function, and can result in a loss of cellular homoeostasis. It is therefore important to understand the chemistry and the downstream biochemical events associated with bioactivation. Information on the chemistry of metabolism coupled with mechanistic biomarkers reflective of certain pathways of hepatic injury would enable both researchers and physicians to predict and diagnose DILI, leading to the improvement of safe drug design. This thesis focuses firstly on the use of in vitro models and mass spectrometry to provide integrated data on the metabolism and toxicity of xenobiotics, using thiophene containing molecules as a paradigm. The thiophene ring has the potential to be bioactivated via S-oxidation and epoxidation pathways, and several thiophene containing drugs have been associated with drug induced liver injury. The investigations described intended firstly to elucidate the chemistry of methapyrilene bioactivation using mass spectrometry and hydrogen-deuterium exchange. The following two chapters aimed to establish a link between bioactivation and toxicity of thiophenes and to evaluate two in vitro models as tool for predicting DILI. The final experimental chapter aims to investigate the potential of ophthalmic acid (OA) to act as a serum biomarker reflective of depletion of hepatic levels of the protective tripeptide, glutathione (GSH). Disturbance of GSH levels through quenching of reactive metabolites can lead to disturbance of its anabolism and catabolism pathways. Indeed, serum OA levels, a GSH analog, have been shown to rise following hepatic GSH depletion. This work utilises GSH adduct formation as a marker of bioactivation of thiophenes in several different in vitro models. Rat liver microsomal incubations were analysed using hydrogen deuterium exchange and LC-MS to define the reactive metabolite of methapyrilene as an S-oxide of the thiophene ring. Freshly isolated rat hepatocytes or single P450 expressing THLE cell cultures were exposed to either methapyrilene, tienilic acid, ticlopidine or 2-phenylthiophene and subsequent LC-MS analysis confirmed GSH adduct formation for all compounds in the isolated rat hepatocyte model, but only for 2-phenylthiophene in the THLE cell model. Cytotoxicity was also investigated in both models, and all compounds were found to cause a greater degree of toxicity in the isolated rat hepatocyte molecule than in the THLE model. By exposing rodents to depletors of hepatic GSH, such as acetaminophen and diethylmaleate, and monitoring the resultant serum OA levels, it has been determined that OA is not a reliable mechanistic marker of hepatic GSH depletion. Kinetic studies of OA in rat serum have revealed that OA is subject to a similar metabolic and elimination pathway as GSH. The overall scope of this work reveals the usefulness of LC-MS/MS to determine S-oxide and epoxide adducts in in vitro studies. The freshly isolated rat hepatocyte model was a useful tool for providing integrated metabolic and toxicological data of thiophene containing molecules and has the potential to be expanded to include data on covalent binding and levels of DILI biomarkers. The single CYP expressing THLE cell model was not as useful in this case, but has been used in other studies to explore the role of discrete P450 enzymes in toxicity and metabolism. Whilst it is unfortunate that serum OA did not reflect hepatic OA in such a way that it could be easily exploited as a biomarker, this does help us to understand that the plethora of potential biomarkers uncovered by proteomic, metabolomic and transcriptomic studies need to be investigated in depth in order to understand their applications across different species and systems.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559389 |
| Date | January 2010 |
| Creators | Walsh, Rachel J. |
| Contributors | Park, Kevin. ; Williams, Dominic |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/1487/ |
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